Bacterial surface proteins have been used as carriers or vehicles of foreign epitopes expressed in the bacterium (particularly in Salmonella and E. coli) for various purposes, including the development of live vaccines. In some instances, the heterologous protein is expressed as a fusion product with a surface protein of the bacterium. Generally, the use of such surface proteins as a vehicle for expression and presentation of heterologous proteins has been limited by the characteristics of the particular surface protein involved. The bacterium's lipopolysaccharide layer, which tends to stimulate a strong immune response, also covers the integral outer membrane proteins of the organism and potentially affects efficient presentation of a cloned epitope. Also, where the surface protein is functional, for example, as part of a filamentous portion of the bacterial cell surface, there will be limited opportunities to express a fusion product and still retain the surface protein's function. Generally, the organisms that have been used for these purposes have been chosen because of the advantages presented in respect of the organism's relationship to its host.
Many genera of bacteria assemble layers composed of repetitive, regularly aligned, proteinaceous sub-units on the outer surface of the cell. These layers are essentially two-dimensional paracrystalline arrays, and being the outer molecular layer of the organism, directly interface with the environment. Such layers are commonly known as S-layers and are found on members of every taxonomic group of walled bacteria including: Archaebacteria; Chlamydia; Cyanobacteria; Acinetobacter; Bacillus; Aquaspirrillum; Caulobacter; Clostridium; Chromatium. (see: Smit, J.; PROTEIN SURFACE LAYERS OF BACTERIA; in: "Offprints From Bacterial Outer Membranes As Model Systems" (1986) Dr. M. Inouye (Ed.); John Wylie and Sons, Inc.).
Typically, an S-layer will be composed of an intricate, geometric array of at least one major protein having a repetitive regular structure. In many cases, such as in Caulobacter, the S-layer protein is synthesized by the cell in large quantities and the S-layer completely envelopes the cell and thus appears to be a protective layer.
Caulobacter bacteria are natural inhabitants of most soil and freshwater environments and may persist in waste water treatment systems and effluents. The bacteria alternate between a stalked cell that is attached to a surface and a motile dispersal cell that has adhesive material already expressed and is searching to find a new surface upon which to stick and convert to a stalked cell. The bacteria attach tenaciously to nearly all surfaces and do so without producing the extracelluar enzymes or polysaccharide "slimes" that are characteristic of most other surface attached bacteria. They have simple requirements for growth. The organism is ubiquitous in the environment and has been isolated from oligotrophic to mesotrophic situations. Caulobacters are known for their ability to tolerate low nutrient level stresses, for example, low phosphate levels. This nutrient can be limiting in many leachate waste streams, especially those with high levels of iron or calcium.
The S-layer of Caulobacter crescentus has been well characterised. Nearly all freshwater isolates of Caulobacter elaborate an S-layer visibly indistinguishable from the one produced by Caulobacter crescentus strains CB2 and CB15. The S-layer proteins from these strains have approximately 100,000 m.w. The protein has been characterized both structurally and chemically. It is composed of ring-like structures spaced at 22 nm intervals arranged in a hexagonal manner on the outer membrane. The S-layer is bound to the bacterial surface by calcium ions and may be removed by low pH treatment or by treatment with a calcium chelator such as EGTA.
The S-layer proteins of S-layer producing strains of Caulobacter have significant similarity. Thus a cloned S-layer protein gene of one Caulobacter strain will likely be useful to retrieve the corresponding genes in other Caulobacter strains (see: Walker, S. G., S. H. Smith, and J. Smit (1992) "Isolation and Comparison of the Paracrystalline Surface Layer Proteins of Freshwater Caulobacters". J. Bacteriol. 174: 1783-1792; and, MacRae, J. O. and, J. Smit (1991) "Characterization of Caulobacters Isolated from Wastewater Treatment Systems" Applied and Environmental Microbiology 57:751-758).
Expression and presentation of a heterologous polypeptide as a fusion product with an S-layer protein of a bacterium would provide advantages not previously seen in systems using organisms such as E. coli and Salmonella where fusion products of other kinds of surface proteins have been expressed. Firstly, many bacteria producing S-layer proteins (particulary Caulobacter) are relatively harmless and ubiquitous in the environment. In contrast, many Salmonella and E. coli strains are pathogens. Consequently, expression and presentation of a heterologous polypeptide using Caulobacter as a vehicle will have the advantage that the expression system will be stable in a variety of outdoor environments and may not present problems associated with the use of a pathogenic organism. Second, many such bacteria, including Caulobacter, are natural biofilm forming species and may be adapted for use in fixed biofilm bioreactors. Finally, the quantity of the S-layer protein that is synthesized by the bacterium and the unique characteristics of the repetitive, two-dimensional S-layer would make such bacteria ideal for use as an expression system and a "presentation surface" for heterologous polypeptides. Such a presentation surface is desirable in a live vaccine so that presentation of a foreign epitope is maximized. In addition, use of the presentation surface to achieve maximal exposure of a desired polypeptide to the environment results in such bacteria being particularly suited for use in bioreactors or as carriers for the polypeptide in aqueous or terrestrial outdoor environments.